Use for raloxifene for the treatment of hepatic cancer

Abstract

The present invention describes novel uses of pharmaceutical compositions comprising raloxifene for the treatment of liver cancer. In human liver cancer cell lines, the compositions of the invention significantly reduced metabolic activity and significantly increased the percentage of apoptotic cells; likewise, they significantly reduced colony-forming capacity. Additionally, the present invention describes for the first time, the novel use of raloxifene for the treatment of liver cancer.

Description

[0001] Use of raloxifene for the treatment of hepatic cancer

[0002] Field of the invention.

[0003] The present invention relates to pharmaceutical compositions useful for the treatment of cancer, specifically to novel uses of pharmaceutical compositions for the treatment of hepatic cancer, more particularly to the use of raloxifene for the effective treatment of hepatic cancer.

[0004] Background of the invention.

[0005] Liver cancer is an aggressive disease with a poor prognosis that frequently occurs in the context of chronic liver disease and cirrhosis. Worldwide, it ranks sixth in the most commonly diagnosed types of cancer and is the third leading cause of cancer death (Sung et al., 2021); according to the WHO, during the year 2020 there were an estimated 905,677 cases worldwide and a mortality of 830,180 in both sexes, being more frequent in men. In Mexico it ranks eighth in incidence and third in mortality; a registry published by the same organization in 2021 reported 7,536 new cases and 7,175 deaths, again being more frequent in the male sex (Ferlay et al. 2021). This represents a worrying mortality-incidence ratio of 95.2%. Like any other cancer, the development of liver cancer involves different factors, among them are genetic and epigenetic alterations, for example, inactivation of tumor suppressor genes and activation of oncogenes, through point mutations, deletions, insertions, etc. (Man et al., 2021). The signaling pathways that are most commonly affected in this type of cancer are Wnt / p-catenin and p53 (Caviglia and Schwabe, 2014). The probability of developing liver cancer is increased when there are chronic inflammatory processes derived from liver diseases such as hepatitis B (HBV) and C (HCV) viral infections, nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH); in fact, at present HBV and HCV infections are the main risk factors in the development of HCC (Sung et al., 2021). On the other hand, harmful chemicals such as excessive alcohol consumption, aflatoxin exposure, and tobacco use have been shown to be contributing factors as well (Massarweh and El-Serag, 2017).

[0006] Unfortunately, liver cancer is considered a silent disease, since at first it does not present signs or attributable symptoms, which makes its diagnosis difficult and unfortunately it is detectable when the patient is in an advanced or intermediate stage, that is, when the tumor already occupies a large part of the organ (Attwa, 2015); at this stage, the most appropriate option is systemic therapy. According to studies in patients with hepatocellular carcinoma (HCC), conventional chemotherapy represents a limitation for the treatment of these patients due to the lack of improvement in survival (Woo and Heo, 2012), that is why a new strategy is needed to combat this disease. As it is known, during the process of carcinogenesis, alteration of various signaling pathways involved occurs, therefore, systemic therapy is directed towards these signaling pathways (Raza, 2014). This type of therapy generally employs multikinase inhibitors such as sorafenib and regorafenib and immune checkpoint inhibitors, employing monoclonal antibodies such as nivolumab (Likhitsup et al., 2019).

[0007] Unfortunately, despite implementing "curative" treatments, it is estimated that the recurrence rate is 15% to 30% in one year and 70% during the next 5 years after treatment (Zhu et al., 2020), so it is clear that work must continue on the implementation of new treatment options. Monotherapy treatment to fight cancer turns out to be ineffective for several reasons, one of them is the development of resistance of cancer cells to available drugs, in addition, conventional treatments using a single molecule are non-selectively targeted to both healthy and cancerous cells (Mokhtari et al., 2017); that is why this disease should be approached from another perspective. Combination therapy has been used for several years now in medicine to treat different diseases, including cancer. This strategy consists of simultaneously administering two or more drugs that are aimed at different cancer-specific drug targets (He et al., 2016). Combination therapy provides great advantages, as it aims to achieve a synergistic effect, in other words, the therapeutic effect of the combination therapy is expected to be greater than the effect of the individual drugs; another advantage is that combination drug dosing regimens reduce the likelihood of generating toxic effects due to the fact that their doses are usually reduced (but allow maintaining pharmacological efficacy); In addition, the fact that this modality of therapy targets different signaling pathways important in cancer decreases the likelihood that cancer cells will develop resistance to treatment (Ayoub, 2021). Chen et al. reported that pimozide, an antipsychotic drug, acts synergistically with sorafenib by enhancing the inhibitory effect on HCC cell proliferation (Chen et al., 2019).

[0008] A retrospective study in patients with HCC demonstrates that combination therapy of lenvatinib with toripalimab, camrelizumab, sintilimab, tislelizumab, or pembrolizumab (monoclonal antibodies targeting programmed cell death protein 1 [PD-1]) had greater effect on improving overall survival, progression-free survival, and time to tumor progression compared with lenvatinib as a single agent (Zhu et al., 2021). In addition, in May 2020 the FDA approved the combination of two monoclonal antibodies, atezolizumab and bevacizumab as treatment for these patients. Additionally, a few years ago through a phase II clinical trial it was shown that the combination of sorafenib with doxorubicin, a conventional chemotherapeutic agent, improved overall survival of patients compared to the chemotherapeutic alone and there was no increase in toxicities (Abou-Alfa et al., 2010). Surprisingly, Zhang et al. reported that disulfiram, a drug used for the treatment of alcoholism, in combination with sorafenib acts synergistically against HCC cells in in vitro and in vivo models (G. Zhang et al., 2022). It is well known that the discovery of new molecules with pharmacological activity is a process with two main disadvantages: time and cost. It takes at least 10 years to bring a new drug to market and the approximate cost is estimated to range from $314 million to $2.8 billion (Wouters et al., 2020). In urgent situations such as cancer, the demand for targeted treatments for this disease is too great, so there is a need for strategies that allow a variety of useful therapeutic options to be obtained in an expedited manner. Thanks to advances in pharmacology and genomics, it has been shown that several non-cancer drugs have great potential to be used for the treatment of multiple types of cancer.

[0009] Drug repurposing is a strategy that consists of the discovery of promising drugs to treat multiple neoplasms from the reuse of drugs whose original use is (or was) intended for other diseases; its main advantage is that it allows an accelerated and lower cost process of obtaining new treatments for cancer, since it is focused on the selection of drugs already approved by the corresponding health institutions, obviously they have already been characterized and their safety in humans is already known (Corsello et al., 2020; Shah and Stonier, 2019). That is why the scientific community has turned its attention to this reuse approach, a reason that has led to the development of strategies for the identification of such molecules in an efficient manner ranging from performing bioinformatics analyses to the application of technologies such as CRISPR-Cas9 (Perez- Plasencia et al., 2021 ; Pushpakom et al., 2019). In this regard, a couple of years ago, a research group developed an innovative tool called PRISM (Profiling Relative Inhibition Simultaneously in Mixtures) that allows rapid testing of many drugs against a large number of cancer cell lines in groups (Corsello et a!., 2020).

[0010] Unfortunately, the diagnosis of liver cancer is made when the tumor has already reached an intermediate or advanced stage in 70-80% of patients. Nowadays, the mortality-incidence ratio is still worrying despite the therapeutic options available, as these prove to be ineffective in advanced stages. Due to the above, there is an urgent need to implement new treatments to combat liver cancer.

[0011] The process of developing new drugs is lengthy and costly, and few molecules are successful in reaching clinical application. The repositioning of non-oncological drugs has offered promising therapeutic alternatives for the treatment of multiple neoplasms. In addition, the combination of drugs improves treatment by achieving a synergistic effect, reducing doses and toxic effects, and minimizing the risk of developing resistance to oncologic drugs. It has been demonstrated that the use of antihistamines and estrogen receptor modulators has antineoplastic activity; therefore, it is necessary to investigate whether their combined application in liver cancer will offer a new useful therapeutic alternative.

[0012] Brief summary of the invention.

[0013] Liver cancer ranks eighth in incidence and third in mortality in our country. Statistics published in 2021 report 7,536 new cases and 7,175 deaths, resulting in a mortality / incidence ratio of 95.2%. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, accounting for approximately 75-85% of cases. The pharmacological therapeutic options available for the treatment of liver cancer in intermediate and advanced stages, which include multikinase inhibitors such as sorafenib and regorafenib, are ineffective, making it necessary to implement new useful therapeutic options. Combination therapy is a strategy that has resulted in obtaining better drug treatments, has synergistic effect, reduces toxic effects and minimizes the likelihood of generating resistance to treatment. In addition, drug repositioning allows obtaining more therapeutic options in an expedited and less costly way compared to the process of discovering new drugs. In this regard, some antihistamines such as loratadine and some estrogen receptor modulators such as raloxifene have been shown to possess antineoplastic activity in both in vitro and in vivo conditions.

[0014] One of the objectives of the present invention is to provide synergistic pharmaceutical compositions for the treatment of liver cancer, for example as a result of implementing the above strategies to evaluate the antineoplastic effect of the combination of the drugs loratadine-raloxifene-sorafenib in human liver cancer cell lines HepG2 and HuH7. The antineoplastic activity of the drugs and their combinations was evaluated by determining their effect on metabolic activity by MTT assay and cell survival by colony formation assay in human liver cancer cell lines HepG2 and HuH7, also the ability of these drugs to induce apoptosis was evaluated using Annexin V-FITC kit. Some of the two- and three-drug combinations resulted in synergistic inhibition of metabolic activity and induction of apoptosis in both cell lines. In HepG2 cells the two- and three-drug combinations inhibited the ability to form colonies. In accordance with the invention, loratadine and raloxifene were shown to possess antineoplastic activity against human liver cancer cells, either alone, in combination and in combination with sorafenib. The invention provides scientific basis for positioning raloxifene, loratadine and their combinations and the combination of the three drugs as innovative treatments for HCC.

[0015] It is another objective of the invention to evaluate the antineoplastic effect of the compositions of the invention comprising the combination of the drugs loratadine- raloxifene-sorafenib, for example in human liver cancer cell lines HepG2 and HuH7.

[0016] It is yet another objective of the invention to determine the inhibitory concentrations 20 (CI20 ) and 50 (CI50 ) of loratadine, raloxifene and sorafenib that are effective in achieving their therapeutic effect, for example by determining the metabolic activity of HepG2 and HuH7 cells by the MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) method.

[0017] It is yet another objective of the invention to evaluate the effect of combinations (CI20 's and CI50 's) of loratadine-sorafenib, raloxifene-sorafenib, loratadine-raloxifene and loratadine-raloxifene- sorafenib on the metabolic activity of HepG2 and HuH7 cell lines by the MTT method.

[0018] It is yet another objective of the invention to determine the effect of the compositions of the invention on the cell death of cancer cells, for example by determining the apoptosis effect of human liver cancer cell lines HepG2 and HuH7.

[0019] It is yet another objective of the invention to determine the effect of the compositions of the invention on the colony formation of human liver cancer cell lines HepG2 and HuH7, as a measure of their antineoplastic effect.

[0020] Brief description of the figures.

[0021] Figure 1. Effect of sorafenib on metabolic activity in HepG2 (upper panel) and HuH7 (lower panel) cell lines. Cells were cultured in the presence of the drug at ten different concentrations (0.5-30 pM) for 72 hours. Results are shown as the mean ± S.D. of eight absorbance readings for each group and represent three different experiments. One-way ANOVA; in both cases significant differences are observed for concentrations of 7.5, 10, 12.5, 15, 20 and 30 pM against the DMSO group (*) according to the Tukey test (P<0.05).

[0022] Figure 2. Effect of raloxifene on metabolic activity in HepG2 (upper panel) and HuH7 (lower panel) cell lines. Cells were cultured in the presence of the drug at ten different concentrations (1-60 pM) for 72 hours. Results are shown as the mean ± S.D. of eight absorbance readings for each group and represent three different experiments. One-way ANOVA; in both cases significant differences are observed for the 20, 25, 30, 40, 50 and 60 pM concentrations against the DMSO group (*) according to the Tukey test (P<0.05).

[0023] Figure 3. Effect of loratadine on metabolic activity in HepG2 (upper panel) and HuH7 (lower panel) cell lines. Cells were cultured in the presence of the drug at ten different concentrations (0.1-50 pM for HepG2 cells and 1-60 pM in HuH7) for 72 hours. Results are shown as the mean ± S.D. of eight absorbance readings for each group and represent three different experiments. One-way ANOVA; significant difference is observed for concentrations of 10, 15, 20, 25, 30, 40, 50 and 60 pM against the DMSO group (*) according to Tukey test (P<0.05).

[0024] Figure 4. Effect of the combination of loratadine-raloxifene, loratadine-sorafenib, raloxifene- sorafenib and loratadine-raloxifene-sorafenib on the metabolic activity of HepG2 cell line, a) Effect of single drugs; b) effect of two-drug combination; c) effect of three-drug combination. The combination of two or three drugs significantly inhibited metabolic activity in both cell lines compared to the effect of single drugs. L= loratadine; R= raloxifene, S= sorafenib. Cells were cultured in the presence of the drug combinations for 72 hours and results are shown as the mean ± S.D. of eight absorbance readings for each group and represent three different experiments. Single-factor ANOVA; significant differences are observed for all treatments against the DMSO group (*); (-) differences against S CI20 ; (♦) differences against S CI50 ; (ft) differences against L CI20 ; (A) differences against L CI50 ; (§) differences against R CI20 and (cp) differences against R CI50 ; (A) differences against S CI20 in the two-combination trial; (0) differences against S CI50 in the two-combination trial according to Tukey test (P<0.05).

[0025] Figure 5. Effect of the combination of loratadine-raloxifene, loratadine-sorafenib, raloxifene- sorafenib and loratadine-raloxifene-sorafenib on the metabolic activity of the HuH7 cell line, a) Effect of single drugs; b) effect of the two-drug combination; c) effect of the three-drug combination. The combination of two or three drugs significantly inhibited metabolic activity in both cell lines compared to the effect of single drugs. L= loratadine; R= raloxifene, S= sorafenib. Cells were cultured in the presence of the drug combinations for 72 hours and results are shown as the mean ± S.D. of eight absorbance readings for each group and represent three different experiments. Single-factor ANOVA; significant differences are observed for all treatments against the DMSO group (*); (-) differences against S CI20 ; (♦) differences against S CI50 ; (ft) differences against L CI20 ; (A) differences against L CI50 ; (§) differences against R CI20 and (cp) differences against R CI50 ; (A) differences against S CI20 in the two-combination trial; (0) differences against S CI50 in the two- combination trial according to Tukey test (P<0.05).

[0026] Figure 6. Effect of the combination of the three drugs on cell morphology in the HepG2 cell line after 72 hours of incubation. Incubation of the cells with the indicated combinations significantly modified cell shape. The red arrow points to the formation of protrusions in a cell. Photographs taken with an Olympus 1X51 10x fluorescence microscope.

[0027] Figure 7. Effect of the combination of the three drugs on cell morphology in the HuH7 cell line after 72 hours of incubation. Incubation of the cells with the indicated combinations significantly modified the cell shape. Photographs taken with an Olympus 1X51 10x fluorescence microscope. Figure 8. Effect of loratadine-raloxifene-sorafenib combinations on apoptosis in the HepG2 cell line. The tested combinations of two and three drugs significantly increased the percentage of cells in apoptosis compared to the activity of individual drugs, with the exception of raloxifene. L= loratadine; R= raloxifene, S= sorafenib. Cells were cultured in the presence of the combinations and individual drugs for 72 hours and results are shown as the mean ± S.D. of readings by flow cytometry employing the annexin V-FITC kit and propidium iodide (PI) staining, representing three different experiments. One-way ANOVA; significant difference is observed in all treatments against the DMSO group (*); (-) differences against S CI20 ; (♦) differences against S CI50 and (A) differences against L CI50 according to the Tukey test (P<0.05).

[0028] Figure 9. Effect of loratadine-raloxifene-sorafenib combinations on apoptosis in the HuH7 cell line. The tested combinations of two and three drugs significantly increased the percentage of cells in apoptosis compared to the activity of individual drugs, with the exception of raloxifene. L= loratadine; R= raloxifene, S= sorafenib. Cells were cultured in the presence of the combinations and individual drugs for 72 hours and results are shown as the mean ± S.D. of readings by flow cytometry employing the annexin V-FITC kit and propidium iodide (PI) staining, representing three different experiments. Single-factor ANOVA; significant difference is observed in all treatments against the DMSO group (*); (-) differences against S CI20 ; (♦) differences against S CI50 ; (A) differences against L CI50 and (cp) differences against R CI50 according to Tukey test (P<0.05).

[0029] Figure 10. Effect of the combination of loratadine, raloxifene and sorafenib on the colony-forming ability of the HepG2 cell line. The combinations tested in this trial were able to inhibit to zero the colony-forming ability of this cell line compared to the effect of the individual drugs. L= loratadine; R= raloxifene, S= sorafenib. Cells were cultured in the presence of the individual drugs and their combinations and DMSO for 72 hours and then allowed to grow for a further 7 days in the absence of the drugs. The results (lower panel) are shown as the mean ± S.D. of readings from three different assays. The upper panel shows photographs of the Petri dishes and shows the colonies stained with crystal violet (1%). One-way ANOVA; significant difference is observed in all treatments against the DMSO group (*); (-) differences against S CI20 according to the Tukey test (P<0.05).

[0030] Figure 11. Integration of the signaling pathways of sorafenib, loratadine and raloxifene involved in the development of HCC. The scheme represents a theoretical proposal of the mechanisms that integrate the activity of the three drugs on relevant molecules involved in the development of HCC where it is observed that they even have signaling pathways in common, which possibly explains the synergistic effect of inhibition of cell proliferation and survival and also promotes apoptosis observed in the present invention.

[0031] Detailed description of the invention.

[0032] The present invention describes and provides synergistic pharmaceutical compositions comprising the combination of at least 2 drugs selected from loratadine, raloxifene and sorafenib, which prove useful for the effective treatment of liver cancer, for example HCC. In the present invention, the compositions described herein, were evaluated for their antineoplastic effect on human liver cancer cell lines HepG2 and HuH7, for example by determining their effect on metabolic activity via MTT assay and cell survival via colony formation assay in the mentioned cell lines; the ability of the combination of these drugs to induce apoptosis was also evaluated employing the Annexin V-FITC kit. Some of the two- and three-drug combinations of the present invention resulted in synergistic inhibition of metabolic activity and induction of apoptosis in both cell lines, while in HepG2 cells the two- and three-drug combinations inhibited the ability to form colonies.

[0033] Pursuant to the present invention, loratadine and raloxifene were shown to possess antineoplastic activity against human liver cancer cells, either alone, and / or in their combination, and / or in their combination with sorafenib. The invention provides scientific basis for positioning raloxifene, loratadine and their combinations, as well as the combination of said combinations with additionally sorafenib as useful combinations for innovative treatments for HCC. In the context of the present invention, it has been shown that the use of antihistamines in patients suffering from some type of cancer such as breast cancer or melanoma is associated with a significant improvement in patient survival; for example, there is strong evidence indicating that astemizole, an H1 antihistamine used for the treatment of allergic rhinitis and conjunctivitis, was associated with a significant reduction in mortality in these patients, so they suggest its evaluation in different cancers (Garcia-Quiroz and Camacho, 2011 ; Shah and Stonier, 2019). Regarding liver cancer, Chavez et. al. evaluated the effect of astemizole on HCC cell lines finding that this drug effectively decreases cell proliferation and increases apoptosis in vitro.

[0034] In addition, other antihistamines such as cyproheptadine (Feng et al., 2015; Hsieh et al., 2017), deptropine (Liang et al., 2020), loratadine and desloratadine (Hu and Carraway, 2020) have also been reported to possess antineoplastic activity.

[0035] Very recently Shen et. al. through cohort studies evaluated the relationship between the use of antihistamines in patients with hepatitis B and / or C virus infection and the risk of developing HCC, finding that the use of antihistamines is associated with a significantly lower risk of HCC (Shen et al., 2022).

[0036] On the other hand, another drug relevant to the present invention belongs to the group of estrogen receptor modulators (SERMs), which have been shown to possess significant antineoplastic activity. Wang et. al. reported the role of raloxifene, a SERM employed in the treatment of osteoporosis in postmenopausal women and breast cancer, on HCC as a potential antineoplastic therapy (Wang et. al., 2017). Likewise, bazedoxifene, another drug classified as a SERM, has been reported to possess antineoplastic activity targeting the same pathway in HCC (Ma et al., 2019; Zafar et al., 2021).

[0037] Therapy aimed at inhibiting various tyrosine kinases is considered the standard treatment for patients with advanced-stage HCC. Among the drugs mostly employed is sorafenib (Formula I), which is an orally administered drug that was approved by the Food and Drug Administration (FDA) in 2007 for the treatment of unresectable HCC and advanced renal cell carcinoma (Abdelgalil et al., 2019). In fact, this multikinase inhibitor was the first drug with this mechanism of action indicated for the treatment of advanced HCC (Eso and Marusawa, 2018). Both serine / threonine and tyrosine kinases play an important role in cellular processes such as proliferation, migration, differentiation, apoptosis, angiogenesis, etc. of malignant cells, which of course possesses an extremely important involvement in cancer development (Eso and Marusawa, 2018).

[0038] Formula I. Chemical structure of Sorafenib

[0039] Therapeutic targets of sorafenib include some serine / threonine and tyrosine kinases such as VEGF, PDGF, RAF-kinases, c-KIT, FLT3, BRAF etc. Inhibition of the activity of these kinases results in decreased angiogenesis, cell proliferation and also induces tumor cell apoptosis (Niu et al., 2021). However, prolonged use of this drug (as of other oncological drugs) increases the risk of cancer cells developing resistance and thus rendering drug treatment ineffective; This is why combination therapies that include sorafenib have been proposed to obtain a better response to treatment, for example, the combination of sorafenib and vorinostat (histone deacetylase inhibitor), temsirolimus (mTOR inhibitor), cixutumumumab (monoclonal antibody) are some examples (Woo and Heo, 2012). Unfortunately, although about 30% of the total number of patients receiving this treatment benefit, after prolonged use (approximately 6 months) they may develop resistance, so that after this period it proves to be ineffective (Tang et al., 2020). Toxicity of sorafenib that has been determined in clinical trials includes the presence of abdominal pain, diarrhea, fatigue, weight loss, hypertension, with skin toxic effects (palmar-plantar erythrodysesthesia and rash) being more frequent, however they can be well controlled (Raoul et al., 2019).

[0040] Raloxifene (Formula II) is a SERM approved by the FDA for the treatment of osteoporosis and is also used for the treatment and prevention of breast cancer (Pinkerton, 2021).

[0041] Formula II. Chemical structure of raloxifene

[0042] In general, raloxifene has a low incidence of adverse effects that are also well tolerated, for example, it increases the risk of venous thromboebolism, although its frequency is less than 1%; hot flashes and leg cramps are also common (Rey et al., 2009). Some SERMs have been found to possess promising antineoplastic activity. As mentioned above, Wang et al. reported that raloxifene is a potent inhibitor of the IL-6 / GP130 signaling pathway, which is involved in the oncogenesis process of various cancers including HCC, such that it could be considered an antineoplastic drug (Wang et al., 2017). This research group observed that raloxifene inhibited cell viability in liver cancer cell lines, including HepG2 and Huh7, and furthermore, through an in vivo model they also demonstrated that it was able to inhibit tumor growth. Additionally, Matsushima et al. reported that both raloxifene and bazedoxifene suppress TGF-a-induced HCC cell migration (Matsushima- Nishiwaki et al., 2022). Such reports provide important evidence positioning this SERM as a potentially useful drug for the treatment of HCC.

[0043] Loratadine (Formula III), a second-generation antihistamine drug, was approved by the FDA for the treatment of allergic rhinitis and urticaria. It is selective for histamine H1 receptors acting as an antagonist, causing decreased vascular permeability, decreased smooth muscle tone, and decreased activation of peripheral nociceptive receptors (Sidhu and Akhondi, 2022).

[0044] Formula III. Chemical structure of loratadine

[0045] In general, loratadine is a drug that is well tolerated by patients, however, some adverse effects may occur, the most common being headache, gastrointestinal discomfort and dizziness, although sedation, mydriasis and xerostomia may also occur but are uncommon (Sidhu and Akhondi, 2022). The use of this antihistamine in patients with some type of cancer has been reported to possess possible antineoplastic effects, for example, Fritz et al. evaluated the effect of loratadine and desloratadine through retrospective studies in patients diagnosed with different types of cancer including breast cancer and melanoma, finding that the use of these antihistamines was associated with improved overall survival (Fritz et al., 2020, 2021). On the other hand, Ellegaard et al. found that the use of loratadine in patients diagnosed with lung cancer was associated with a significant reduction in mortality (Ellegaard et al., 2016). Interestingly, they found that such mortality reduction was greater in patients with a history of chemotherapy. Regarding HCC, the cytotoxic effect of the combination between loratadine and cisplatin was evaluated in HCC cell lines, finding that both drugs act synergistically (Adly, 2018), however, until before the present invention the effect of loratadine as such in liver cancer had not been reported.

[0046] The present invention has as its main purpose to provide synergistic pharmaceutical compositions comprising appropriate (optimal) combinations of at least two of the compounds of formula I and / or formula II and / or formula III that produce high antineoplastic efficacy (synergism) with lower doses (generating lower probability of causing adverse effects), which are extremely useful for the treatment of liver cancer, for example HCC.

[0047] It is one of the modalities of the invention to provide pharmaceutical compositions comprising a combination of at least 5 pM and / or of at least 25 pM and / or of at least 10 pM of the compounds of formula I and / or formula II and / or formula III respectively, in order to obtain antineoplastic effects of high or maximum efficacy, thus significantly reducing the necessary doses of said compounds in combination. The treating physician can adjust the unit dose, as well as the maximum daily dose, which has an optimized antineoplastic effect described, and which he / she considers to be the best, depending on the circumstances of each patient. It is another modality of the invention, the possibility of using the pharmaceutical compositions described herein, preferably comprising from 4.04 to 8.6 pM and / or from 19.6 to 25.2 pM and / or from 10.5 to 30 pM of the compounds of formula I and / or formula II and / or formula III respectively and pharmaceutically acceptable derivative salts thereof, to prepare a medicament for the effective and efficient treatment of liver cancer, for example HCC, with the lowest possible doses.

[0048] It is another embodiment of the invention to use pharmaceutical compositions comprising synergistic combinations of the compounds of formula I and / or formula II and / or formula III suitable to be administered subcutaneously, orally, intraperitoneally, intraperitoneally, intramuscularly, intravenously or topically, in doses that are synergistically effective for the treatment of liver cancer, for example HCC.

[0049] It is another of the modalities of the invention, the possibility of formulating pharmaceutical forms comprising as active principles the compounds of formula I and / or formula II and / or formula III or their pharmaceutically acceptable salts to make them administrable by different routes to the patient who requires it, being a mammal including the human, and being able to be adapted to provide the unitary doses that allow exerting the antineoplastic effect described in the treatment of hepatic cancer, where the pharmaceutical forms are obtained by a person of the technical field of pharmacology without being limited to any specific one, and seeking to preserve the efficacy of the drugs in terms of the synergistic effect described, either by releasing the active ingredient quickly or slowly, or instead seeking greater efficiency in the target tissue, avoiding damage to the patient by chemical interaction, solubilizing insoluble substances, improving flavors, improving aspects and facilitating the doctor the posology to perform the treatment, being the different existing pharmaceutical forms solid, semisolid or liquid, for example: powders (which may be encapsulated), granules and tablets or tablets, pills, and suppositories. Also its administration in patches and in semi-solid pharmaceutical forms: ointments, pastes, gels, or liquid pharmaceutical forms: solutions, injections, syrups, suspensions or eye drops.

[0050] Examples of pharmaceutically acceptable excipients accompanying the active principles of the invention are for example, for oral administration as tablets or tablets, agents comprising for example, diluents, binders, stabilizers, bulking agents, thickening agents, such as povidone, microcrystalline cellulose, lactose, etc., disintegrating agents such as for example cross-linked carboxymethyl cellulose, surfactants such as for example sodium lauryl sulfate, lubricating or sliding agents such as for example magnesium stearate, colloidal silicon dioxide, etc., wherein said excipients may be formulated for preferably slow or prolonged release for a systemic effect.

[0051] Solutions for intravenous or intraperitoneal administration can be prepared from the active ingredients dissolved first in an organic solvent such as DMSO, ethanol, or dimethylformamide and subsequently in aqueous buffers, such as PBS.

[0052] Particular preference is given to dosage forms designed for systemic administration, wherein liquid or solid compositions can be formulated, suitable for administration and whose excipients can be selected for example from components compatible with said dosage form, for example of a lipidic or peptidic nature, or peptidomimetics, known in the prior art as non-immunogenic, and which preferably can be bound to the active principles of the invention to improve their bioavailability; propellant agents such as, for example, propane, butane, or permissible chlorofluorocarbons; pH regulators such as, for example, sulfuric acid; chelating agents such as, for example, EDTA. The active ingredients can also be formed into micronized particles contained in gelatin capsules or other systems known in the technical field, which help to release the active ingredient to its target site of action, for example, by means of solid dosage forms such as tablets or dragees, including those formulated for prolonged release.

[0053] According to the present invention, the synergistic compositions described herein may be obtained by combining the compounds of formula I and / or formula II and / or formula III or their pharmaceutically acceptable salts with pharmaceutically compatible and / or acceptable vehicles known in the art, in the amounts and / or concentrations that correspond as described herein, and compounds known in the art may be included to obtain such compositions. Likewise, the administration of such compositions, may be made depending on the conditions of the patient, which will determine the doses and frequency of administration necessary to achieve an effective treatment of the ailment each particular case, although it is preferred to dose the compositions of the present invention according to the concentrations and dosage described herein.

[0054] Another purpose of the invention is to provide novel uses of raloxifene (compound of formula II) for the treatment of hepatic cancer, such as for example HCC. In this sense, the use of pharmaceutical compositions comprising at least 25 pM of raloxifene, allows to observe its antineoplastic effects of high or maximum efficacy, for which the treating physician can adjust the unit dose, as well as the maximum dose per day, which has an optimized antineoplastic effect described, and which he considers to be the best, depending on the circumstances of each patient. Likewise, in an additional modality of the invention, the use of pharmaceutical compositions comprising from 19.6 to 25.2 pM of raloxifene for the treatment of hepatic cancer is further preferred.

[0055] Still another purpose of the invention is to provide novel uses of loratadine (compound of formula III) for the treatment of hepatic cancer, such as for example HCC. In this sense, the use of pharmaceutical compositions comprising at least 10 pM of loratadine, allows to observe its antineoplastic effects of high or maximum efficacy, for which the treating physician can adjust the unit dose, as well as the maximum dose per day, which has an optimized antineoplastic effect described, and which he considers to be the best, depending on the circumstances of each patient. Likewise, in an additional embodiment of the invention, the use of pharmaceutical compositions comprising from 10.5 to 30 pM of loratadine for the treatment of hepatic cancer is further preferred.

[0056] In accordance with the present invention, we demonstrate that both loratadine and raloxifene have significant effects on the inhibition of metabolic activity in both cell lines (HepG2 and HuH7) from 10 pM and 20 pM respectively.

[0057] The concentrations of raloxifene determined in the present invention that were shown to have significant effect on cell proliferation, cell survival and induction of apoptosis were, on average, 20.4 pM and 24.6 pM (CI20 and CI50 respectively), which is above the maximum concentrations reached by raloxifene determined in healthy males after oral administration of 60 mg / day, which is the indicated dose for the treatment of osteoporosis and corresponds to 0.00063 pM (0.33 ng / mL) (Lee et al., 2022), and in postmenopausal women with the same dose a maximum blood concentration of 0.001 pM (0.5 ng / mL) is reported (Hochner-Celnikier, 1999). Furthermore, reported therapeutic concentrations range from 0.001-0.002 pM (0.67-1.3 ng / mL) (Schulz et al., 2020). So far, no clinical trials have been conducted to demonstrate the concentrations that can achieve an antineoplastic effect in humans, however, some clinical trials have been conducted in which patients were administered daily doses of up to 600 mg / day for 8 weeks and 120 mg / day for 3 years without serious toxicities (Draper et al., 2009), only leg cramps and dizziness were reported. Additionally, toxicity studies in animal models have indicated that raloxifene at high doses is well tolerated; single doses of up to 5000 mg / kg have been administered in rodents (rats and mice) and 1000 mg / kg in monkeys (Evista product insert).

[0058] On the other hand, loratadine reaches plasma concentrations of 0.00004 pM (17 pg / L) in healthy volunteers (Zhang et al., 2003). The therapeutic dose of loratadine ranges from 0.0026-0.052 pM (0.001-0.02 mg / L) (Schulz et al., 2020). The CI20 and CI50 on average determined in the present invention were 11.65 pM and 26.5 pM respectively, indicating that they were higher than the bioavailable concentrations achieved. Like raloxifene, no clinical trials have been conducted to determine the dose at which loratadine is capable of producing antineoplastic effect in humans, but there is clinical evidence indicating that both raloxifene and loratadine have been shown to have positive effects on improving overall patient survival and reducing mortality when administered at their indicated doses for the treatment of osteoporosis and as an antihistamine (60 and 10 mg / day respectively).

[0059] Loratadine is an antihistamine with great safety for administration in patients, however, it was reported that ingestion of a single 300 mg dose of loratadine in a 6-year-old child resulted in a slight increase in blood pressure and heart rate (Cobb et al., 2001).

[0060] Morphologically, both loratadine and raloxifene in combination and alone caused visible changes in cell structure suggesting that the cells might be undergoing apoptosis (Zhang et al., 2018).

[0061] Sorafenib is an oncology drug targeting tyrosine kinases, among the main ones it inhibits we have multiple receptor tyrosine kinases (RTKs) such as VEGFR (1 , 2 and 3), PDGFR, KIT, FLT3 and FGFR1 (Gong et al., 2017), in addition, it is also known to be able to inhibit the RAS / RAF / MEK / ERK pathway, which has an extremely important role in cell proliferation, differentiation and survival and is overactivated in many types of cancer (McCubrey et al., 2007). Thus, blocking the activity of these kinases leads to inhibition of important processes in cancer progression such as cell proliferation, apoptosis and angiogenesis.

[0062] One of the reasons we focused our attention on raloxifene is because there is evidence that estradiol has a protective role against liver damage and prevents the development of HOC, and it is even thought to be a factor in why the incidence of liver cancer is higher in men than in premenopausal women (Shi et al., 2014; Q. Wei et al., 2015). In addition, estrogen deficiency in peri- and postmenopausal women is thought to lead to an increased risk of developing liver damage (Brady, 2015). To date, several targets of raloxifene involved in the development of HCC have been identified and the possible mechanisms that explain the activity observed in the present invention are discussed below. First, raloxifene, being a SERM, has affinity for both ER isoforms and depending on the tissue in which they are expressed, it can act as an agonist or antagonist. It is believed that the tissue-specific expression of both isoforms and their dimerization conformations induced by SERMs affect tissue-specific activity, thus in breast and uterus, raloxifene acts as an antagonist, while in bone and liver it has agonist activity (Martinkovich et al., 2014). Both ER-a and ER-p are expressed in the liver under normal conditions but their expression is modified during inflammatory processes. Both isoforms are decreased in HCC patients compared to healthy tissue samples (Sukocheva, 2018) and are thought to lose their function during disease progression, even the ER-a isoform could be considered a predictor of poor prognosis of HCC (Shi et al., 2014). It is well known that nuclear factor KB (NF-KB), in Kupffer cells, stimulates the production of proinflammatory cytokines, one of the most relevant in the development of HCC is interleukin 6 (IL-6), as when this is produced in excess it has a strong influence on liver carcinogenesis (Xu et al., 2021). Naugler et al. reported on the possible protective capacity of estrogen in inducing liver injury in male mice that were administered diethylnitrosamine (DEN), observing that estrogen inhibited IL-6 secretion and in turn significantly reduced DEN-induced injury (Naugler et al., 2007). When IL-6 binds to its receptor, it recruits Janus Kinase (JAK) which, through downstream signaling activation, promotes phosphorylation and in turn activation of STAT3, a transcription factor involved in cancer development through activation of proliferative processes, angiogenesis, invasion, etc. (Svinka et al., 2014). In addition, ER-a can directly interact with NF-KB and inhibit IL-6 secretion, raloxifene could interact with ER-a and inhibit IL-6 secretion and thus tumor progression. On the other hand, among the ER-a target genes we have the protein tyrosine phosphatase receptor type O (PTPRO), a membrane protein that has tumor suppressor activity in various types of cancer, including HCC (Xie et al., 2021). One of its functions is tyrosine dephosphorylation and its targets include JAK2 and PI3K. Possibly raloxifene could be activating ER-a, which in turn favors the expression of PTPRO and the following inhibition of the mentioned kinases, causing then the inhibition of STAT3 and the subsequent inhibition of cancer cell growth and survival (Meng and Liu, 2022). Not only ER-a participates in the protective capacity of estrogen, but there is also evidence that the ER-p isoform is involved. Matsushima et al. reported the involvement of raloxifene and bazedoxifene in inhibiting HCC progression through their specific interaction with ER-p. They propose that both drugs could be activating the said receptor in the liver, which, through downstream signal transduction, inhibits Akt activity and as a result inhibits cell migration (Matsushima et al., 2022). Sun et al. provided relevant information on the involvement of the PI3K / Akt / mTOR signaling pathway in the mechanism of cell survival and proliferation, angiogenesis and apoptosis, processes that are deregulated during HCC (Sun et al., 2021). When a ligand binds to its RTK, they recruit phosphoinositide 3-kinase (PI3K) which subsequently phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) resulting in phosphatidylinositol-3,4,5-trisphosphate (PIP3), which can recruit protein kinase B (Akt) to the membrane who is responsible for activating downstream proteins such as the mammalian cell target of rapamycin (mTOR). One of the functions of mTOR is to regulate cell growth, proliferation and survival. In addition, another target of Akt is the transcription factor CREB, whose target genes are also involved in the process of proliferation and apoptosis in many types of cancer including HOC.

[0063] Knowing this, perhaps one of the mechanisms that could explain the observed activity of raloxifene in the present invention is through the activation of both ER isoforms that are expressed in the liver cancer cells tested. Firstly, the canonical pathway of activation of the nuclear estrogen receptor consists of binding to its ligand and subsequent translocation to the cell nucleus and binding to sequences known as estrogen response elements (EREs) and thus, the subsequent transcription of target genes (Fuentes and Silveyra, 2019), which would be favoring the aforementioned mechanisms. It was recently described that estrogen not only has activity through the participation of its nuclear receptors, but there is also a receptor that has non-genomic activity, i.e. , the G-protein- coupled transmembrane estrogen receptor (GPER). The role of this receptor seems to be inconsistent depending on the type of cancer in question, for example, in breast cancer, GPER is involved in progression and metastasis and its expression is associated with poor prognosis, it has even been associated with tamoxifen resistance (Lappano et al., 2014). A tumor progression effect has also been described in ovarian, endometrial, prostate and lung cancer related to its activity (Hernandez-Silva et al., 2020; Xu et al., 2019). In contrast, in gastric cancers, activation of GPER inhibits cell proliferation and induces apoptosis (Lee et al., 2019). In HCC, the involvement of this receptor has not yet been fully investigated, but Qiu et al. suggest that GPER activation in this cancer could represent a therapeutic target. They reported that activation of this receptor in HCCLM3 and SMMC-7721 liver cancer cell lines led to sustained activation of the ERK pathway, a fact that surprisingly resulted in decreased cell viability and induction of apoptosis, while in mouse xenograft models, the GPER / ERK pathway significantly suppressed tumor growth (Qiu et al., 2021). This finding is curious because the ERK signaling pathway is associated with cancer progression (Kohno and Pouyssegur, 2006), however, there is evidence to support that sustained ERK activation can lead to cell death (Lau et al., 2017). Wei et al. suggest that GPER activation exerts a protective effect against tumorogenesis in HCC through modulation of the inflammatory response. Using a DEN-induced mouse model of HCC, they found that silencing GPER expression significantly increased liver tumorogenesis, furthermore they found that the expression level of this receptor is higher in non-tumor tissue compared to tumor tissue in human HCC samples (Wei et al., 2016). Thus, this constitutes another possible mechanism by which raloxifene could be exerting the activity observed in the present invention.

[0064] Raloxifene has the ability to bind to both ER isoforms, however, it has a higher affinity for ER-a than for ER-p, presenting binding affinities (Ki) of 0.52 and 20.2 nmol / L respectively (Moen and Keating, 2008), so it could be thought that the observed activity is mostly due to the participation, firstly, of the a isoform, subsequently the p and finally of GPER, since the latter is weakly expressed in HepG2 and HuH7 cell lines (Wei et al., 2016). Interestingly, raloxifene would not only be exerting the activity observed in the present invention through ER activation, as Wang et al. found that this drug exerts pharmacological activity even in the absence of these receptors. This research group supports the fact that raloxifene would be inhibiting the release of IL-6 but they propose that it does so through the impairment of the formation of the IL-6 / IL- 6R / GP130 complex (Wang et al., 2017), which is essential for this cytokine to initiate the downstream signal transcription process and promote the inflammatory process and cancer development (Reeh et al., 2019).

[0065] Another pharmacological target of raloxifene that has been identified, independently of its estrogenic action, is the aryl hydrocarbon receptor (AhR). O'Donnell et al reported the involvement of this receptor in triple-negative breast cancer cells. They described that its activation inhibits cell proliferation and induces apoptosis in MDA-MB-231 cells, furthermore, they identified raloxifene as a ligand of this receptor and tested its activity in human hepatoma cell lines in which it induced apoptosis (O'Donnell et al., 2014). Knowing this, possibly raloxifene is exerting its agonist activity on this receptor, since HepG2 and HuH7 cell lines also express it, and in this way it favors the inhibition of cell proliferation and survival and the induction of apoptosis.

[0066] However, some antihistamines have attracted the attention of physicians and pharmacists to be used in the therapy of different cancers. We have the case of Fritz et al., who reported that the use of some antihistamines, including loratadine, was associated with a significant improvement in the overall survival of patients consuming them who were diagnosed with breast cancer and melanoma (Fritz et al., 2020). In the present invention, loratadine was also shown to have inhibitory activity on cell proliferation and survival, in addition it was able to induce apoptosis in HepG2 and HuH7 cell lines. The possible mechanisms involved in this activity, in the first place, could be due to its main role as an antagonist of the histamine type 1 receptor (H1 HR) as a recent study points out that there is an overexpression of this receptor in HCC tumor tissues and its presence is related to cancer progression and poor prognosis through cell cycle induction, matrix metallopeptidase 2 (MMP2) production and suppression of apoptosis. Zhao et al. reported that inhibiting this receptor significantly suppressed tumor growth and metastasis in a mouse xenograft model of HCC (Zhao et al., 2020).

[0067] It seems that the participation of TRPV2, a calcium channel that is expressed and related to several types of cancer, is involved in the progression of HCC, since it has been reported that the levels of both mRNA and protein of this channel are increased in well-differentiated HCC tumor tissue compared to undifferentiated tissue, and there is a strong relationship between its expression and invasion into the portal vein. Van den Eynde et al. reported that TRPV2 is related to endometrial cancer progression and identified loratadine, astemizole and clemizole as TRPV2 blockers, with loratadine being the most potent antagonist, leading to inhibition of cell proliferation and migration in in vitro assays in HEK293 cells (Van den Eynde et al., 2022). However, there is still no evidence to show that loratadine has similar effects in HCC, possibly this antihistamine is directed towards this channel in HepG2 and HuH2 cells, which resulted in the inhibition of cell proliferation and survival observed here, however, research is lacking to confirm this. Ellegaard et al. indicate that it is possible to take advantage of changes in lysosomal behavior related to cancer, especially the strong sensitization of the lysosomal membrane that allows the entry of anticancer drugs that can cause cell death, as is the case of cationic amphiphilic drugs (CAD) (Ellegaard et al., 2016; Kallunki et al., 2013). One of these drugs is precisely loratadine, which could be exerting the observed activity through such mechanism, but again, to date there are no records of works that have investigated this possibility. Moreover, it has been described that loratadine can exert anti-inflammatory activity by inhibiting the inflammatory response triggered by NF-KB signaling (Hunto et al., 2020), an effect that reduces the levels of proinflammatory components, including IL-6 and TNF-a. In addition, loratadine can suppress the activator protein 1 (AP-1) signaling pathway, a transcription factor whose activation enables the transcription of inflammation-related enzymes including MMPs (Jang et al., 2022); it is also involved in proliferation, survival, differentiation, apoptosis and cell migration (Ye et al., 2014); in fact, very recently it was described that MMPs promote the progression of HCC and are also significantly increased in these tissues, so they could function as a biomarker of poor prognosis in patients (L. Xu et al., 2022). For this reason, loratadine is probably exerting the antineoplastic activity observed here through its anti-inflammatory function.

[0068] In the present invention we observed that when combining sorafenib with loratadine or with raloxifene, in both cell lines there was a decrease in metabolic activity, which is commonly interpreted as an index of cell proliferation, an effect that was concentration dependent. Interestingly, the combination of loratadine with raloxifene had a greater effect on the inhibition of cell proliferation compared to the combination of sorafenib with either of the two, a fact that was not expected, since being an oncological drug, it was expected that its effect would result in an increase in antiproliferative activity when combined with either of the two, even the combination of the ICS20 , according to the Cl analysis, resulted in an antagonistic effect. On the other hand, the combination of the ICS20 and IC50 of the three drugs resulted in a very potent inhibition of the metabolic activity of both cell lines with respect to the effect of the individual drugs or the combination of two of them, and the type of interaction that prevailed was synergism, according to the Cl determination. It was also observed that the combination of the three drugs caused a significant increase in the induction of apoptosis in the two cell lines compared to the effect of the individual drugs or the combinations of two of them, with the exception of raloxifene, which surprisingly strongly induced apoptosis similar to the effect caused by the combinations evaluated. It is important to note that the combinations described here increased the percentage of cells in late apoptosis in the HepG2 cell line, while in HuH7 it increased the percentage in early apoptosis. This fact could indicate that HuH7 cells present a slower mechanism of apoptosis or are less sensitive to the effect of drugs in this process. Such differences between results could also be due to some other factors such as the nature of the cell lines, i.e., while the HuH7 line originates from an HCC tumor, the HepG2 line corresponds to a hepatoblastoma tumor, which gives each line different molecular characteristics that could be involved in such results. In order to know with certainty which characteristics give each line greater or lesser sensitivity to the effect of the drugs, it is necessary to carry out relevant assays to determine this, for example, the use of genomic and proteomic studies. Finally, the combination of the three drugs according to the present invention also proved to have a significantly greater effect on the inhibition of cell survival compared to the individual activity of each drug, since the combinations tested were able to decrease to zero the number of colonies formed, an effect that was also observed with the IC50 of raloxifene.

[0069] The possible mechanisms that explain the synergistic effect observed in the present invention could be justified because the three drugs share activity on several signaling pathways, for example, both sorafenib and raloxifene could be simultaneously inhibiting the PI3K / Akt / mTOR pathway, favoring together the inhibition of cell proliferation and survival in a concentration-dependent manner, however, the Cl determination showed that when combining the CI20 of both drugs an antagonistic effect occurred, this could be explained because possibly raloxifene is saturating the targets to which sorafenib is directed, causing a decrease in its effect. In addition, it is important to take into account that raloxifene could be inducing over-activation of the ERK pathway as mentioned above and, on the other hand, one of the mechanisms of sorafenib is to inhibit this pathway, so that at this point the activity of both drugs incubated simultaneously is contradictory and possibly reflected in the antagonistic effect observed. In the case of loratadine, relevant research is still needed to elucidate a more accurate mechanism to clarify the observed activity; however, its participation in the inhibition of IL-6 secretion converges with the mechanism described for raloxifene, which could be participating in the observed synergistic effect. Likewise, the Ras / MEK / ERK pathway favors AP-1 activity (Chang et al., 2003) but, as it is also being inhibited by sorafenib, there will be a decrease in AP-1 , adding the participation of loratadine on this transcription factor could possibly explain the synergistic effect observed. On the other hand, loratadine could be inhibiting the expression of MMPs through two mechanisms, through AP-1 inhibition and H1 HR antagonism.

[0070] The fact that loratadine targets different molecules compared to the other two drugs favors the fact that together they manage to inhibit cell proliferation and survival and also the induction of apoptosis. Additionally, the combination of the three drugs strongly inhibited the aforementioned activities in both cell lines, this can be explained because the three drugs, in addition to converging on different signaling pathways, are known to be directed to different targets that are important in the development of HCC (Figure 11).

[0071] In the present invention we demonstrated that the use of loratadine and raloxifene were able to significantly inhibit cell viability and survival, as well as induce apoptosis under in vitro conditions in liver cancer cells. There is strong preclinical as well as clinical evidence that positions them as potential candidates to become new treatment options against liver cancer. In addition, we showed for the first time that the combination of the three drugs tested was effective in causing inhibition of proliferation and viability of cultured liver cancer cells, as well as in inducing apoptosis. We also demonstrate that the combination of the three drugs provides an advantage in achieving greater chances of targeting cancer cells, in this case in the liver, and that reusing drugs that have already been approved and are indicated for other diseases are useful for targeting cancer.

[0072] According to the present invention, raloxifene, in comparison with loratadine and sorafenib, was the drug that was shown to have the greatest activity employed individually on the induction of apoptosis and inhibition of colony formation in the liver cancer cells evaluated. Likewise, the combination of sorafenib with raloxifene and / or loratadine inhibited cell proliferation to a greater extent in the HepG2 and HuH7 cell lines, compared to the effect of each drug tested individually, also increased the percentage of cells in late apoptosis in the HepG2 cell line and the percentage in early apoptosis in the HuH7 line, and further inhibited cell survival by inhibiting the colony-forming ability of HepG2 cells compared to the effect of each drug tested individually.

[0073] The following examples are included below for the sole purpose of illustrating the invention and its modalities, without implying limitations to its scope.

[0074] Example 1. Materials and methods.

[0075] - Cell lines and reagents. The human liver cancer cell lines used were HepG2 and HuH7, both obtained from the American Type Culture Collection (ATCC, Manassas, VA). They were cultured according to the manufacturer's instructions. In all assays the cells were seeded and cultured for 24 hours and then exposed to the different drugs. The drugs (sorafenib, raloxifene and loratadine) were purchased from Sigma Aldrich. In all assays the concentration of vehicle (DMSO) was less than 0.06%.

[0076] - Determination of inhibitory concentrations (IC) and drug interactions. The ICS20 and ICsso were determined by analyzing the concentration-response curves of the three drugs obtained by MTT assays. The inhibitory concentration is defined as the concentration reguired to inhibit cellular metabolic activity at 20% and 50% of the maximum effect. The determination of drug interactions was performed through the calculation of the combination index (Cl) by applying the Chou-Talalay mathematical model indicated below, which indicates that if Cl <1 , the interaction between two or more drugs is of the synergistic type; if Cl = 1 then it is of the additive type, while if Cl >1 , it is considered antagonistic (Chou, 2010). where,

[0077] D1 = concentration of drug 1 used,

[0078] D2 = used concentration of drug 2,

[0079] Dm1 = inhibitory concentration 50 of drug 1 ,

[0080] Dm2 = inhibitory concentration 50 of drug 2, fa = affected fraction of cell proliferation when the two drugs are co-incubated (normalized / 100), ml = slope obtained from the concentration-response curve of drug 1 , m2 = slope obtained from the drug concentration-response curve 2.

[0081] - Metabolic activity. Metabolic activity was assessed using the MTT method. HepG2 and HuH7 cells were seeded in 96-well plates (4,000 cells x well) and incubated for 72 hours at 37°C in culture medium alone, in the presence of the drugs (loratadine, raloxifene, sorafenib, and their combinations (CI20 's and CI50 ’s)) or in the presence of vehicle (DMSO). MTT was added 4 hours before the end of the incubation period, then SDS was added to the wells and left to incubate overnight. The next day the absorbances were read with a ThermoFisher Multiskan SkyHigh microplate spectrophotometer, using two filters (595 nm and 690 nm).

[0082] - Apoptosis. Apoptosis was determined using the Annexin V-FITC kit (Thermo Fisher Scientific, Waltham, MA, USA). HepG2 and HuH7 cells were seeded in 60 mm Petri dishes (50,000 cells x plate) for 72 hours at 37°C in culture medium alone, in the presence of the individual drugs and selected combinations or vehicle (DMSO). Camptothecin (apoptosis inducer) and methanol (necrosis inducer) were used as controls, which were added to the cells 24 hr before the end of the incubation period. The samples were then read on the CYAN ADP cytometer flow cytometer (Dako, Glostrup, Denmark). Cells were sorted as follows: viable (FITC-negative and Pl-negative), early apoptotic (FITC-positive and Pl-negative), late apoptotic (FITC-positive and Pl-positive) and necrotic (FITC-negative and Pl-positive), the percentages of which were obtained by quadrant analysis using Kaluza version 2.1 software.

[0083] - Colony formation. This assay allowed measurement of cell survival. HepG2 cells were seeded in 60 mm Petri dishes (400 cells x plate) isolated from each other to allow colony growth from single cells. Twenty-four hours later, the cells were incubated for 72 hours at 37°C in culture medium alone in the presence of the individual drugs and selected combinations or vehicle (DMSO). Subsequently, in the absence of the drugs, the cells were allowed to grow for an additional 7 days. After this time, the cells were fixed with ethanol (absolute grade) for 15 minutes and stained with 1 % crystal violet again for 15 minutes, washed with water and counted.

[0084] - Statistical analysis. Statistical analysis of the data was performed using GraphPad Prism software version 8.0 by single-factor ANOVA analysis, followed by Tukey's test. Differences were considered statistically significant when p<0.05.

[0085] Example 2. Determination of the ICS20 and ICso of sorafenib, raloxifene and loratadine in human liver cancer cell lines. The analyses of the concentration-response curves of the three drugs to obtain the IC's used in the subsequent experiments are shown below. All experiments included a control group (cells incubated only with culture medium during the 72 hours of the experiment).

[0086] Sorafenib. Figure 1 shows the effect of this drug on normalized metabolic activity in both cell lines (HepG2 and HuH7). Ten different concentrations were evaluated and compared against the vehicle condition (DMSO); in both cell lines statistically significant differences were observed at 5 pM and above in the effect of the drug on metabolic activity. The IC values2o and IC50 are given in Table 1. Raloxifene. As with sorafenib, the effect of raloxifene on metabolic activity was evaluated in both cell lines at 10 different concentrations observing that from 25 pM onwards there were statistically significant differences. Raloxifene inhibited metabolic activity in both cell lines in a dose-dependent manner (Figure 2), with respect to the DMSO group.

[0087] Loratadine. The effect of this third drug was also evaluated on metabolic activity in both cell lines at the concentrations shown in Figure 3, observing significant differences in the effect of the drug on metabolic activity from 10 pM in both cases. The values for both ICs are shown in Table 1 .

[0088] Example 3. The combination of loratadine-raloxifene, loratadine-sorafenib, raloxifene- sorafenib and loratadine-raloxifene-sorafenib exhibit strong anti-metabolic activity in HepG2 and HuH7 cell lines. Figure 4 shows the quantitative determination of the effect of the combination of two and three drugs in HepG2 cells on metabolic activity and HuH7 (Figure 5). Panel a) of each figure shows the effect of the individual drugs, panel b) shows the effect of the combination of the two-drug IC20 and two-drug IC50 on the corresponding cell line, while panel c) shows the effect of the combination of the three-drug ICs. Surprisingly, the combination of the ICS20 of sorafenib with loratadine or with raloxifene presented an antagonistic effect that was not expected (Table 2), however, when co-incubating the IC50 of any of these two drugs with the IC20 and IC50 of sorafenib, a synergistic effect occurred according to the determination of the combination index (Cl) (Table 2). Some of the two-drug combinations resulted in a significant inhibition (compared against the effect of the DMSO group and against that of each individual drug) of metabolic activity in both cell lines, an effect that was increased when co-incubated with the three-drug combinations, where more than 70% of the inhibition of metabolic activity was observed with respect to the DMSO group and, furthermore, the effect of some three-drug combinations was statistically different from their combination with two drugs (Figure 4 and 5).

[0089] Table 1. Inhibitory concentrations CI20 and CI50 of sorafenib, raloxifene and loratadine in HepG2 and HuH7 cell lines.

[0090] *pM

[0091] Table 2 shows the determinations of the combination index (Cl) calculated using the equation proposed by Chou-Talalay, which provides information on the type of interaction between combinations of two or more drugs. In most cases, the effect of co-incubating two or three drugs was synergistic, although there were combinations that, according to this parameter, presented an antagonistic effect such as sorafenib with any of the ICS20 the two drugs and which was also more frequent in HuH7 cells, in which there was also an additive effect.

[0092] Figures 6 and 7 show the cell shape of both cell lines after they were exposed for 72 hours to the indicated drug combinations. In the first instance, comparing the effect of the three combinations illustrated against the DMSO group, it is observed that the cells lost the interaction between them, in addition, they showed a round shape, the bright color of the cells was lost and they showed a dark coloration and in some cases it was possible to observe the formation of protrusions in the cell membranes.

[0093] From the metabolic activity experiments, the combinations used for the subsequent experiments were selected. The following combinations were tested in both cell lines: L CI50 + R CI50 , S CI20 + L CI20 + R CI20 and S CI50 + L CI20 + R CI50 , additionally, the combination S CI50 + L CI20 + R CI50 was tested in HuH7 cells. Table 2. Determination of the combination index (Cl).

[0094] Combination Cl Interaction type

[0095] Selected combinations that were tested in apoptosis and colony formation assays are indicated in bold. Sorafenib (S), Raloxifen (R), Loratadine (L).

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Claims

Claims.1 . A pharmaceutical composition for use in the treatment of liver cancer, characterized in that it comprises Raloxifene.

2. The pharmaceutical composition for use according to claim 1 , characterized in that the therapeutically effective amount of Raloxifene is at least 25 pM.

3. The pharmaceutical composition according to claim 2, characterized in that the therapeutically effective amount of Raloxifene is from 19.6 to 25.2 pM.

4. The pharmaceutical composition according to claim 3, characterized in that the therapeutically effective amount of Raloxifene is from 20.4 to 24.6 pM.

5. The use of Raloxifene for the manufacture of a drug for the treatment of liver cancer.

6. The use in accordance with claim 5, wherein the cancer is hepatocellular cancer (HCC).

7. The use in accordance with claim 5 to 6, wherein the raloxifene is administrable in an amount of at least 25 pM.

8. The use in accordance with claim 7, wherein raloxifene is administrable in an amount of 19.6 to 25.2 pM.

9. The use in accordance with claim 8, wherein the raloxifene is administrable in an amount of20.4 to 24.6 pM

Images